Secondary Structure in Enzyme‐Inspired Polymer Catalysts Impacts Water Oxidation Efficiency

Abstract Protein structure plays an essential role on their stability, functionality, and catalytic activity. In this work, the interplay between the β‐sheet structure and its catalytic implications to the design of enzyme‐inspired materials is investigated. Here, inspiration is drawn from the active sites and β‐sheet rich structure of the highly efficient multicopper oxidase (MCO) to engineer a bio‐inspired electrocatalyst for water oxidation utilizing the abundant metal, copper. Copper ions are coordinated to poly‐histidine (polyCuHis), as they are in MCO active sites. The resultant polyCuHis material effectively promotes water oxidation with low overpotentials (0.15 V) in alkaline systems. This activity is due to the 3D structure of the poly‐histidine backbone. By increasing the prevalence of β‐sheet structure and decreasing the random coil nature of the polyCuHis secondary structures, this study is able to modulates the electrocatalytic activity of this material is modulated, shifting it toward water oxidation. These results highlight the crucial role of the local environment at catalytic sites for efficient, energy‐relevant transformations. Moreover, this work highlights the importance of conformational structure in the design of scaffolds for high‐performance electrocatalysts.


S1.2. Electrochemical measurements
Electrochemical measurements were performed using a Gamry 600 Reference potentiostat (Gamry Instruments, Warminster, PA, USA), using Pt wire and an Ag/AgCl electrode (3 M NaCl filling solution) as counter and reference electrodes respectively, and glassy carbon (GC), carbon cloth (CC), or highly oriented pyrolytic graphite (HOPG) as working electrode.The potential values were converted to values against the standard hydrogen electrode (SHE) using the formula   / 0.21, where ESHE is the potential value in Volts versus SHE and EAg/AgCl is the measured potential value in Volts versus the Ag/AgCl electrode.All potential values are reported against SHE.
The measured currents were converted to current densities by normalizing to the geometric area of the working electrode.All measurements were performed at ambient temperature.
Prior to electrochemical measurement, GC was cleaned by polishing with a 0.05 μm alumina slurry, followed by sonication in isopropanol and deionized water for 5 minutes.CC was sonicated in 70% (v/v) ethanol and water for 10 min.HOPG was cleaned using the scotch tape method.A piece of tape was pressed onto the HOPG surface and then pulled off to remove the top layers of the HOPG.The edge-plane surfaces were isolated with nitrocellulose resin, to expose only the HOPG clean basalplane surface to the electrolyte.

S1.3. Copper-histidine complex synthesis and electropolymerization
Copper-histidine complex Cu(His)2 was prepared by mixing Cu 2+ and histidine at the 1:2 molar ratio.For that, 20 μL of 1.0 M CuSO4 solution in ultrapure water was added to 200 μL of 0.2 M L-histidine solution previously prepared in 0.1 M sodium phosphate solution (pH 9.0).The final concentration of Cu(His)2 was 91.0 mM.The color of CuSO4 solution changes from light to dark blue when added to L-histidine solution, indicating complexation.The featured band at 640 nm in the UV-visible spectrum solution confirms the Cu(His)2 formation (Figure S1). [19]For electropolymerization on the electrode surface, a suitable volume of freshly-prepared Cu(His)2 complex solution was added to 0.1 M sodium phosphate solution (pH 9.0) to a final concentration of 20.0 mM.A potential was then applied between -1.01 to 2.21 V at 100 mV s -1 for 25 cycles to induce polymerization, as previously described. [20]Subsequently, the electrode was rinsed three times with ultrafiltered water.

S1.4. Micro-FTIR spectroscopy
Spectroscopic measurements in the infrared region (IR) were performed in a Vertex 70v FTIR spectrometer (Bruker, Germany).The spectra were collected in the reflectance mode between 4,000 and 1,300 cm -1 at 64 accumulations with a 4 cm -1 resolution in six different regions of each sample.Optical images were collected with a 36× objective lens.For the micro-FTIR spectroscopic measurements, polyCuHis was electropolymerized on an HOPG surface.The samples were rinsed three times with ultrafiltered water and dried under vacuum for at least 30 min prior to analysis.

S1.5. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS)
The measurements were recorded using a Zeiss Merlin High-resolution scanning electron microscope (Jena, Germany) with an accelerating voltage of 16 kV.PolyCuHis was electropolymerized on a CC surface as described in Section S1.3.The samples were rinsed in ultrapure water and dried under vacuum overnight before the measurements.Samples were attached on a typical pin stub using carbon conducting tape and silver paste prior to the analyses.Wavelength / nm S1.6.X-ray photoelectron spectroscopy (XPS) XPS measurements were performed at Brazilian Nanotechnology National Laboratory (LNNano), part of the Brazilian Centre for Research in Energy and Materials (CNPEM).A Thermo Scientific K-Alpha X-ray photoelectron spectrometer with a monochromatic Al Kα X-ray (hν = 1486 eV) excitation source was used.All spectra were measured using a spot size of 300 µm and 10 scans.The XPS survey spectrum was recorded at pass energy of 200.0 eV, energy step size of 1.00 eV, and dwell time of 10 ms.The high-resolution spectra at C 1s, N 1s, O 1s, and Cu 2p regions were recorded at pass energy of 50.0 eV, energy step size of 0.10 eV, and dwell time of 50 ms.PolyCuHis was electropolymerized on carbon cloth and rinsed three times with ultrafiltered water and dried under vacuum for at least 30 min prior to analysis.S1.6.Inductively coupled plasma mass spectrometry (ICP-MS) An Agilent 7900 IC-PMS (serial # JP15180835) was used to measure copper ( 63 Cu).The samples were measured with helium gas, and germanium ( 72 Ge) was used as an internal standard.Test and calibration samples (3.0 mL) in 0.1 M sodium phosphate solution (pH 9.0) were lyophilized and then resuspended and incubated in 1.5 mL 60% HNO3 overnight.Next, they were diluted in ultrapure water with 2% HNO3 and filtered through a 0.22 µm filter prior analyses.Count / a.u.
Binding energy / eV XPS survey spectrum (Figure S3a) shows the presence of Cu, N, O, and C atoms, expected to polyCuHis electropolymerized on carbon cloth.Cu/N atomic ratio was calculated being 0.23, which suggests that one copper ion is coordinated for 1-2 histidine residues.These insights into the coordination environment enable optimization of the scaffold to enhance electrocatalysis.
High-resolution spectrum in Cu 2p region shows the copper characteristic peaks at 933.4 and 953.2 eV (Figure S3b), which are features of Cu(II) 2p3/2 and Cu(II) 2p1/2.The satellite peak at 944 eV indicated that the chelation of copper in the form of Cu(II). [21,22]Cu(I) compounds in contrast to paramagnetic Cu(II) compounds do not show satellites in Cu 2p spectra. [22]The spectrum profile is very different from that expected for copper oxides and copper hydroxide, [23] indicating that those species are not formed on the electropolymerization process.The deconvolution of highresolution spectrum in N 1s region (Figure S3c) displays two peaks, at 401.2 eV, which are assigned to Cu-N, [24] and at 399.9 eV, attributed to amide (N-C=O), amine (C-NR2, where R is C and/or H), and C-N=C groups. [24,25]This result evidences the presence of Cu coordinated for histidine by N atoms.The deconvoluted high-resolution spectrum in O 1s region (Figure S3d) also shows two peaks are assigned to carbonyl (C=O, 531.9 eV) and carboxylic (-COOH, 533.7 eV) groups.[27] The functional groups observed in the oxygen and carbon high-resolution spectra can be attributed to histidine and/or groups on the carbon fibers where the polyCuHis was electropolymerized.For a further investigation about the copper coordination to histidine in polyCuHis, FTIR measurements were performed and compared to standard histidine (Figure S4).The involvement of the amine group and water molecules in coordination can be supported by the analysis of the higher wavenumber region of the spectrum (Figure S4a), although the band broadening due to Hbonds made the assignments difficult.The band at 3267 cm -1 in the histidine spectrum, attributed to the symmetric streaching of NH2 group, [28] is suppressed in the polyCuHis spectrum, possibly indicating the formation of amidic bound and coordination with Cu(II).The appearance of a band at 3433 cm -1 in polyCuHis spectrum (Figure S4b) is assigned to O-H stretching, and suggests the coordination of H2O molecules to Cu(II). [28]In addition, the band at 1440 cm -1 in the histidine spectrum, featured of and CN stretching and NH(ring) and CH2 in-plane bending modes, [10,11] are suppressed in the polyCuHis spectrum, indicating the coordination of Cu(II) to the N atoms from histidine imidazole group.The signal at 1384 cm -1 , attributed to the COO -symmetric stretching, [30,31] do not change in histidine and polyCuHis spectra, revealing that the histidine carboxylic groups are not involved in the copper ion coordination.The copper ion coordination to N atoms is also supported by changes in the vibrational modes at smaller wavenumbers (Figure S4c).The signals at 970 cm -1 , assigned to NH(ring) out of plane bending, CN stretching, CHx and imidazole in plane bending modes, [28,29] and at 926 cm -1 , corresponding to N3C4 stretching, [32] shift to smaller wavenumbers (940 and 906 cm -1 , respectively), indicating coordination to the copper ion.

S3. Copper-histidine complex for water oxidation
The small molecule copper-histidine complex (Cu(His)2) was investigated as a homogeneous electrocatalyst for the WOR.Cyclic voltammograms were recorded with GC in 0.1 M sodium phosphate solution (pH 9.0) containing 10.0 mM Cu(His)2 (Figure S5).The onset potential for the WOR oxidation is 0.95 V.

S4. Electropolymerization of polyHis
PolyHis in the absence of coordinated Cu 2+ was electropolymerized on GC surfaces through the application of 25 successive potential cycles between -1.01 and 2.21 V at 100 mV s -1 in 0.1 M sodium phosphate solution (pH 9.0) containing 20.0 mM His monomer (Figure S6).The oxidation peak at 1.48 V in the initial voltametric curve is related to the histidine monomer oxidation.The decrease in the oxidation peak currents and the peak potential shift to more positive values over the successive potential cycles indicate the histidine polymerization on the electrode surface. [20,33]

S6. Varying the total polyCuHis amount
PolyCuHis was electropolymerized on GC surfaces by 10 successive potential cycles between -1.01 and 2.21 V at 100 mV s -1 in 0.1 M sodium phosphate solution (pH 9.0) containing 20.0 mM Cu(His)2.The electrode was subsequently rinsed three times with ultrapure water, followed by transfer to fresh 0.1 M sodium phosphate solution (pH 9.0) for WOR evaluation (Figure S7).

S7. Butler-Volmer model
The Butler-Volmer equation (Equation S1) determines the current at a specific overpotential (E-E o´) in the absence of mass-transfer impacts or limitations.

𝑖 𝑖 e
´ e ´ S1 where, i is current, i0 is the exchange current (balanced faradaic anodic or cathodic current at equilibrium), α is the transfer coefficient, F and R are the Faraday and gas constants, respectively, E is the applied potential and E 0´ the formal potential.For The Butler-Volmer curve was simulated to fit the experimental voltametric data at low potentials (0.70-1.25 V).

S8. Electrode surface area and polyCuHis on CC electrode
The electrochemical surface area of the carbon electrodes was determined by cyclic voltammetry.Quantitatively, larger capacitive current (capacitive current = anodic currentcathodic current) indicates higher electrical double layer capacitance of the electrode and is a reliable indicator of electrochemical surface area.Representative cyclic voltammograms of GC and CC obtained in 0.1 M sodium phosphate electrolyte solution (pH 9.0) in a non-faradic potential region are displayed in Figure S8.The capacitive current densities of GC and CC at 0.40 V are 0.10 ± 0.01 and 0.37 ± 0.02 mA cm -2 , respectively.This indicates that the electrochemical surface area of CC is higher than that of GC by a factor of 3.7.

Figure S8
. Cyclic voltammograms of CC and GC at 100 mV s -1 in 0.1 M sodium phosphate solution (pH 9.0).
PolyCuHis was electropolymerized on CC electrode as described in Section S1.3.Constant potential measurements at 1.21 V (Figure S9) show the steady-state current for WOR with this polymer to be 0.105 mA cm -2 (red), whereas the bare CC (blank, grey) shows an oxidative current of 0.044 mA cm -2 .

S9. Electrochemical detection of the produced O 2
To detect the generated O2 from the WOR catalyzed by polyCuHis, classical amperometric sensor based on the oxygen reduction reaction on Pt was used. [37,38]The O2 detection is based on the O2 reduction on a Pt wire polarized at -0.30 V (vs SHE), by current monitoring with time.The sensor was coupled to the electrochemical cell where water is catalytically oxidized by polyCuHis polarized at 1.21 V (same potential used in the TON calculation) for in situ O2 detection.The system was calibrated in the presence and absence of O2 dissolved in the electrolyte (phosphate solution, pH 9.0).The response curve (blue curve in Figure S10) shows the electroreduction of the produced O2 a few seconds after 1.21 V is applied to the polyCuHis-modified CC (at 100 s, indicated by ON in Figure S10).The increase in the current density to more negative values (from 0.05 mA cm -2 at 100 s to -0.23 mA cm -2 at 690 s) indicates the electroreduction of the O2 produced by the polyCuHis.As soon as the WOR potential application was interrupted (indicated by OFF in Figure S10), the measured O2 reduction current decreases.A control amperometric curve was recorded applying 1.21 V (vs SHE) at an unmodified carbon cloth electrode (Figure S10, grey curve).

S10. Enzymatic colorimetric H 2 O 2 detection
As H2O2 is not the desired WOR product but can be produced as an electrolysis by-product, we investigated whether this chemical was produced in our system.H2O2 quantification was performed in triplicate using horseradish peroxidase (HRP) reaction-driven 2,2'-azinobis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS) color development (see equation S2).
PolyCuHis electropolymerized on a CC electrode (0.7 cm 2 ) was employed for water electrolysis at 1.21 V for 600 s in 2.5 mL of 0.1 M sodium phosphate solution (pH 9.0), as shown in Figure S11.The oxidation charge was calculated by the integration of each curve to be 0.110, 0.112, and 0.091 C for electrodes #1, #2, and #3, respectively.The molar concentration of H2O2 produced considering 100% of current efficiency as calculated by equation S3 to be 1.35 ± 0.12 mM.

𝑐 𝑄 𝑛F𝑉 S3
where,  is the H2O2 concentration (mol L -1 ), Q is the charge (C) obtained from the integration of the current-time curves, n is de number of electrons involved in the reaction (2 electrons, in this case), F is Faraday constant (9,485 C mol -1 ), and V is reaction volume (0.0025 L, in this case).The effective H2O2 concentration produced by electrolysis can be determined by enzymatic assay with HRP enzyme and ABTS (equation S3).For that, a calibration curve with standard H2O2 (2-75 µM H2O2) was built (Figure S12a).The linear fit generated an equation  0.1208 0.0224, with R 2 equals to 0.9996.
After the electrolysis, the electrolyte pH was adjusted to 5.0 by addition of suitable volume (approximately 20 µL) of 37% HCl.In a 96-well plate, 188 µL of each sample was added to a freshly prepared 2 µL of 0.05 µg mL -1 HRP (Thermo Scientific, prod.no.21134) and 2 µL of 20 mM ABTS (final concentration = 1 mM) in 0.1 M phosphate buffer (pH 5.0).After 15 min of reaction, the absorbance at 410 nm of three electrolyte samples was measured at 25 o C, as well as the absorbance of a blank solution (1 mM ABTS in 0.1 M phosphate buffer, pH 5.0), as control (Figure S12b).No considerable absorbance change was observed compared to the control solutions, indicating that no significant H2O2 was found in the electrolyte samples.

S11. Determination of copper ion release following the application of an electrochemical potential
To determine whether copper ions are released from the polymer upon an applied potential for WOR, ICP-MS was used.A copper calibration curve was generated, and the electrolyte was collected following application of 1.21 V for 600 s at the polyCuHis-modified electrode (Figure S13b, samples #1-3).A blank sample was prepared in fresh electrolyte as a control.Samples were prepared as described in Section S1.6.Additionally, a freshly polymerized electrode was incubated in 60% HNO3 overnight to dissolve the polyCuHis and the solution was analyzed for total Cu determination on the electrode (Figure S9b, sample #4).Copper quantification was accomplished through the generation of a calibration curve (Figure S13a).The linear fit generated an equation  7979.54.855 10 , with R 2 = 0.998.Copper was then quantified following subtraction of the blank scan from each sample.Concentration was then corrected to incorporate subtraction of the blank from each sample following correction for the dilution factor employed in the sample preparation.Concentration was then corrected to incorporate dilution factor.Only negligible copper was found in the electrolyte (samples #1-3) following the application of an electrochemical potential, suggesting that copper is not released from the modified electrode.The total Cu content on the electrode (sample #4) was calculated to be 1.24 × 10 -7 mol cm -2 .Amide A (ν(NH)) [39]   3067 Amide B (ν(NH)) [39]   2915-2850 ν s /ν as (CH 2 ) [40]   1643 Amide I (ν(C=O)) [40]   1533 Amide II (ν(CN) and ν(CH)) [39]   1458 His, δ as (CH 3 ), δ(CH 2 ), ν(CN) [40]   1394 ν s (COO -) [40]   ν: stretching; νs: symmetric stretching; νas: asymmetric stretching; δ: bending; δ as: asymmetric bending.The surface excess (Γ) of copper sites on the electrode surface was calculated according to Equations S4 based on the copper anodic peak charge (Equation S5 and Figure S17) in the first cycle of electropolymerization.
where, Q is the charge of anodic peak (C), n is the number of electrons, F is Faraday constant (96,485 C mol -1 ) and A is the geometric area of the electrode (cm 2 ), i is the faradaic current, υ is the scan rate (V s -1 ), and E is the applied potential (V).Γ of copper was determined to be 5.95 × 10 -5 mol cm 2 .

Figure S17.
The anodic curve of the first electropolymerization cycle after subtraction of the capacitive current.
The amount of water oxidized per second (mol s -1 ) was calculated based on the chronoamperogram at 1.21 V.The amount of water oxidized per second (mol s -1 ) was then normalized by the total number of copper sites on the electrode surface to determine the TOF, the number of water molecules oxidized per Cu 2+ site per second (s -1 ).

S2.
Figure S2.(a) SEM image of polyCuHis film and (b) its EDX spectrum indicating the presence of carbon, oxygen, nitrogen copper and chlorine.(c) Carbon, (d) oxygen, (e) nitrogen, and (f) copper map in image (a).

Figure S3 .
Figure S3.(a) XPS survey spectrum of polyCuHis film.High resolution spectra in (b) Cu 2p, (c) N 1s, (d) O 1s, and (e) C 1s regions, where solid black lines represent raw spectra, wine, blue, green, orange, and pink solid lines are the deconvoluted peaks and dashed red lines are the sums of the deconvoluted peaks.

Figure S4 .
Figure S4.Zoomed views of the FTIR spectra of polyCuHis (blue curves) and standard histidine (black curves) in (a) high, (b) middle, and (c)small wavenumber regions.

Figure S5 .
Figure S5.(a) Voltammograms at 50 mV s -1 recorded with GC electrode in 0.1 M sodium phosphate electrolyte solution (pH 9.0).Scans were obtained in the absence (grey) and presence (red) of 10 mM Cu(His)2.(b) Zoomed view of the voltammogram showing the onset potential for the WOR.

Figure S10 .
Figure S10.Chronoamperometry curves recorded with the Pt-based O2 sensor while 1.21 V is applied to polyCuHis on CC (blue) and bare CC (grey).

Figure S11 .
Figure S11.Chronoamperometry curves at 1.21 V recorded with CC electrodes modified with polyCuHis.

Figure S12 .
Figure S12.(a) H2O2 calibration curve.(b) Absorbance value at 410 nm of blank solution (1 mM ABTS in 0.1 M phosphate buffer, pH 5.0) and the electrolyte after electrolysis with polyCuHis on CC electrode.

Figure S13 .
Figure S13.(a) Copper calibration curve by ICP-MS and (b) copper signal measured in each sample and in the blank solution.

Figure S15 .
Figure S15.Zoomed view of amide I band (black line) of charge-induced polyCuHis, the deconvoluted curves (filled curves), and their sum (dashed red line).

Figure S16 .
Figure S16.Changes in the amide I band of polyCuHis with the charge induction.

Table S1 .
Electrochemical performance of reported copper-based complexes for WOR catalysis.